Abstract: All-trans retinoic acid (atRA) is an active metabolite of vitamin A that acts through retinoic acid receptors (RARs) to regulate gene expression. Increasing studies report RAR-independent (non-canonical) activity of atRA signaling in various biological systems. Importantly, genetic studies have established that cellular retinoic acid binding protein 1 (Crabp1) mediates this atRA non-canonical activity, such as rapid modulation of extracellular signal?regulated kinase (ERK) activation in stem cell cycle control. Recently, we have identified another signaling target of Crabp1, calcium (Ca2+)-calmodulin dependent protein kinase II (CaMKII). CaMKII is an important kinase implicated in numerous cellular functions. Preliminary data in this proposal support the physiological relevance of the Crabp1-CaMKII signalsome in mediating the action of vitamin A as a nutrient and an endocrine that modulates cellular signaling capacity. Our preliminary data reveal dramatically altered CaMKII activity in Crabp1 knockout (CKO) mouse motor neurons. Human genetic data identified significantly reduced Crabp1 expression in certain motor neuron diseases, further supporting the physiological and pathological relevance for Crabp1. The principal hypothesis is that the Crabp1-signalsome orchestrates a novel (RAR- independent) atRA-responsive mechanism that tightly regulates activation of important intracellular signaling components such as CaMKII and ERK. I hypothesize that this Crabp1-CaMKII signalsome (the focus of this proposal) plays a role in modulating motor neuron activity. The goal of my proposal is to elucidate the mechanism of this Crabp1-CamKII signalsome. To achieve this goal, two aims are proposed. Aim 1 will characterize the exact molecular relationship between Crabp1 and CaMKII using molecular, biochemical, and structural biology approaches. The effect of atRA and compounds from our library (Crabp1-selective and RAR-independent) on this molecular relationship will also be determined. Aim 2 will functionally validate Crabp1 in regulating CaMKII activity in a physiologically relevant cellular context of Ca2+-regulated neuronal functions. This will be accomplished by employing a) reconstituted neuron cell lines to molecularly dissect critical elements required for CaMKII signaling and b) viral rescue of Crabp1 expression in primary CKO motor neurons, followed by functional validation of this Crabp1-CaMKII pathway. Functional assays include 1) phosphorylation of CaMKII substrates AMPAR, CREB, and synapsin. 2) Measurement of intracellular Ca2+ concentrations that mediate neuron activity/firing (a known output of CaMKII activity). The significance of this proposal is to provide the first molecular understanding of a new non-canonical signaling of vitamin A/atRA that modulates CaMKII activity and plays a role in protecting against certain human diseases. In a nutrition/endocrine context, this proposal addresses a new cross-talk between atRA (an endocrine and essential nutrient) and an important Ca2+ signaling molecule, CaMKII. The results will provide insight on the increasingly recognized complexity in vitamin A signaling and the development of novel retinoid therapeutics for human disease.